Recombinant DNA stems from the discovery of enzymes called restriction endonucleases or “restriction enzymes,” that are capable of cleaving double stranded DNA at specific recognition sites, thereby producing DNA fragments that can be ligated to one another by ligase enzyme to generate “recombinant” molecules (see, for example, Cohen et al, Proc. Natl. Acad. Sci. USA 70:1293, 1973; Cohen et al., Proc. Natl. Acad. Sci. USA 70:3274, 1972; see also U.S. Pat. Nos. 4,740,470; 4,468,464; 4,237,224). The study of molecular biology has benefited greatly from the ability of scientists to join DNA fragments together in manmade arrangements for the purpose of experimentation or industrial production.
Molecular biology was further facilitated by the invention of the polymerase chain reaction (PCR), which allowed rapid in vitro amplification of selected DNA segments. This allowed for production of large amounts of replicated material that could subsequently be cleaved by restriction enzymes and ligated to other DNA molecules (see, for example, U.S. Pat. Nos. 4,638,195; 4,683,202; 5,333,675). Further advances of the PCR technique included the creation of a DNA polymerase having enhanced thermostability and polymerase mixtures having enhanced fidelity and length of product (U.S. Pat. No. 5,436,149). PCR products can be cloned using restriction enzymes, if the primers are made longer to incorporate the desired restriction site, if said site is not present in the PCR product or cutting at it can be suppressed, and if the vector has a similar matching restriction site. It is important to carry out a purification of the PCR product to at least remove dNTPs and/or polymerase before digestion by restriction enzymes, especially for 5′-sticky ends, since they can be filled in by said left-over DNA polymerase.
A way to clone PCR products without restriction enzymes, and also using RNA/DNA primers, has been recently described [Chen G J., Qiu N. & Page MGP (2002) Universal restriction site-free cloning method using chimeric primers, Biotechniques 32:518-524]. This method still uses ligase, and the ribo base cleavage is not enzymatic, but rather by the use of rare-earth metal ions, which are not as efficient, convenient, or specific as RNAse enzyme.
Ways to clone the PCR products without the use of ligase have been developed. For instance, TOPO (™Invitrogen)—cloning utilizes special vectors with adjacent sites for Vaccinia virus topoisomerase, and said vector is pre-activated with topoisomerase. If the PCR product has terminal extra As, and no 5′-phosphates, it can be cloned into this vector conveniently, at a site between the two topoisomerase recognition sequences. The present invention has no requirement for any specific vector or even the site of cloning within any vector, except that the vector must preferably be amplifiable by PCR using primers at the cloning site. Another example of a non-ligase cloning method utilizes terminal homology similar to the present invention, although usually twice as long. Special E. coli strains which highly express the recET system, or any yeast strain (if the vector is a yeast vector) can then be used to recombine target and vector. This system is not very efficient, and is prone to recombination and rearrangements at other homologous or repeated sites on the vector and/or target molecules, rather than just at the desired and small terminal homologies.
Thus despite these advances, there is substantial room for improvement. The use of restriction enzymes requires that the segment of DNA being digested have the particular restriction site only at desired locations—that is to say, a particular DNA base sequence (restriction site) is necessary to enable the restriction enzyme to digest the particular piece of DNA, yet the restriction sites must be rare or preferably non-existent within the vector or target, lest they be cut up too much to put together in the desired arrangement. Often, 2 vector or 2 target molecules will join, often in the wrong orientation, whereas the desired product is usually one vector and one target with one desired orientation.
Therefore, molecular biology could benefit from the development of improved systems of cloning and joining of DNA molecules. Of particular interest would be a system that would allow DNA joining without the use of, or indeed any regard for, restriction enzymes or restriction sites, provide for efficient joining, provide an increase in yield and specificity of the desired product, decrease the cost of molecular biological experiments, and be generally useful for the joining of DNA sequences having a wide variety of sequences. Optimal systems would even provide for directional joining (i.e., joining in which the DNA molecules to be linked together will only link to one another in a single orientation).